Identification of vacuolar proteins delivered by the AP-3 pathway. (a) Model highlighting the analysis of the AP-3 pathway by deletion of the AP-3 subunit APL5. (b) Volcano plot identifying cargoes that are enriched or depleted at vacuoles of apl5Δ cells. Analysis was performed as in Fig. 3 b. Known AP-3 cargo proteins are color coded in green. The two insets represent magnifications of the dense areas in close proximity to the hyperbolic curve. (c) Localization of Ego3-GFP in Vph1-mCherry–stained WT cells (upper panel) and apl5Δ cells (lower panel). Scale bar, 5 µm. (d) AP-3 mutants are hypersensitive to rapamycin treatment. WT, apl5Δ, apl6Δ, apm3Δ, aps3Δ, and ego3Δ cells were spotted on control plates or plates containing 4 ng/ml rapamycin. (e) Localization of N-terminal GFP-tagged Vac7 in FM4-64 WT cells (upper panel) and apl5Δ cells (lower panel). Scale bar, 5 µm. (f) Localization of N-terminal mCherry-tagged Ypk9 in WT (left panel) and apl5∆ (right panel) cells. The vacuolar lumen was stained with CMAC. Scale bar, 5 µm. (f and g) Superplot showing the quantification of images in f from three different experiments (error bars = SD). Single experiments are color coded and the average is shown as the black line. (h) Experimental setup for the visual genetics screen. Vph1-mKate–expressing WT cells, apl6Δ cells, and vps45Δ cells were crossed against a library of mutants expressing N-terminally GFP-tagged proteins annotated to localize to the vacuole, sporulated, selected for haploids, and imaged with an automated microscope. (h and i) N-terminally GFP-tagged Syg1- and Vph1-mKate–expressing WT cells (upper panel), vps45Δ cells (middle panel), and apl6Δ cells (lower panel) are shown as an example from the screen performed in h. Scale bar, 5 µM.